This invention relates generally to biochemical compounds, and in particular to caged compounds. More specifically, the invention relates to caged NADP and NAD.
The pyridine nucleotides nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are the most abundant coenzymes in eukaryotic cells and have long been studied as oxidative cofactors. These pyridine nucleotides (referred to herein as "the pyridine nucleotide coenzymes") serve as cofactors for all known dehydrogenases and many reductases and hydroxylases, making them the major carriers of protons (H.sup.+) and electrons (e.sup.-) in a number of major metabolic systems, including the glycolysis pathway, the tricarboxylic acid cycle, fatty acid synthesis, and sterol synthesis. NAD and NADP generally have distinct physiological roles. Oxidation of NADH to NAD releases an electron pair which ultimately results in the formation of ATP, making NAD the major cellular energy source for oxidative phosphorylation. NADP, on the other hand, plays a central role in moving electrons along reductive cellular metabolic pathways.
The pyridine nucleotide coenzymes also have an assortment of non-redox functions. For example, NAD is the substrate for poly (ADP-ribose) synthetase, which adds poly ADP-ribose to chromatin and other nuclear proteins in eukaryotic cell nuclei. In this function NAD plays an important role in DNA repair, cell differentiation and DNA synthesis. NAD also serves as the substrate the mono ADP-rihosylation of host proteins by pathogenic bacteria and for the poly ADP-ribosylation of chromatin. Both NAD and NADP are involved in the phagosomal production of oxygen radicals in neutrophils, and are precursors to compounds involved in a complex process of regulating calcium (Ca.sup.+2) mobilization.
The importance of the pyridine nucleotide coenzymes is reflected in the extraordinary number of analogs which have been synthesized to study their various functions. To date, approximately 200 NADP and NAD analogs have been constructed, including fluorescent derivatives, structural analogs, affinity labels, and spin-labeled analogs. In addition, dozens of derivatives with substituted or altered nicotinamide have been constructed and used to investigate the redox potential of hydride transfer and to study coenzyme binding sites. However, no successful "caged" NAD and NADP compounds are known.
"Caged" compounds are biologically inactive molecules designed to fragment into biologically active molecules upon photolysis. The strategy of adding photolabile groups to biomolecules has been an invaluable resource since Hoffmann and coworkers introduced caged ATP in 1978 (Kaplan, J. H., Forbush, B. d., and Hoffman, J. F. (1978) Biochemistry 17, 1929-1935). A review of known caged compounds is provided by Corrie, J. E. T. and Trentham, D. R. (1993) in Bioorganic Photochemistry Volume 2: Biological Applications of Photochemical Switches (Morrison, H., Ed.) pp 243-305, John Wiley & Sons, Inc., New York, which is incorporated herein by reference for all purposes. As inert compounds, caged molecules can be introduced into biological systems over relatively long periods and then be activated rapidly by light, providing a means of exerting temporal and spatial control over the introduction of physiologically active compounds into complex systems. This approach has proven invaluable in systems where traditional methods of rapid mixing are not possible, such as with membrane preparations, living cells, tissue slices, and protein crystals.
Caged compounds are typically constructed by covalently modifying a biologically active molecule with a photolabile protecting, or "caging", group. Given the function of caged compounds, the key characteristics of an effective caging group are its ability to render the substrate inert in the particular system being studied, and to then release it rapidly and efficiently following photolysis. It is also advantageous if the caging group has an absorbance spectrum different enough from the compound being caged so that photolysis interference is avoided, and if it is unreactive with surrounding proteins when released.
One important application of caged compounds is in determining the structures of short-lived enzymatic intermediates using time-resolved Laue crystallography. This technique is described, for example, in Moffat, K., Szebenyi, D., and Bilderback, D. (1984) Science 223, 1423-1425, which is incorporated by reference herein for all purposes. Irradiation of protein crystals soaked with caged compounds can rapidly initiate synchronous turnover within the crystal, allowing catalytic intermediates to be visualized crystallographically. For example, caged GTP has been used to characterize the GTPase activity of Ha-ras p21 protein, as described in Schlichting, I., Almo, S. C., Rapp, G., Wilson, K., Petratos, K., Lentfer, A., Wittinghofer, A., Kabsch, W., Pai, E. F., Petsko, G. A., and Goody, R. S. (1990) Nature 345, 309-315.
Accordingly, caged NADP and NAD compounds would be desirable in many areas of biomedical research. Moreover, the ability to design caged NAD/P compounds with biochemically distinct activities could be advantageous for some applications of these compounds.